An alkaline phosphatase protection assay to investigate trp repressor/operator interactions.

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al.

Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA.

We have employed a filter binding assay to help study the mechanism by which bound L-tryptophan enables the Escherichia coli trp repressor to bind its operators. We have prepared variants of the trp repressor using structural analogues of the natural corepressor, L-tryptophan, and measured the affinity of these variants for a 20-base pair oligonucleotide duplex containing a symmetrical idealization of the trp operator from the E. coli trpEDCBA operon.

Crystal structure of trp repressor/operator complex at atomic resolution.

The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface.

6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin.

The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished.

The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor.

We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor. By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor. We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor.

Crystals of the trp repressor-operator complex suitable for X-ray diffraction analysis.

Crystals of a simulated trp repressor-operator complex have been grown that are large enough and are sufficiently well ordered and durable to provide a high quality molecular image of this regulatory protein X DNA complex to better than 3-A resolution. The "operator" consists of a 2-fold rotationally symmetric 18-base pair duplex that is extended by a dT residue at both 5'-termini. This system exhibits extensive crystal polymorphism.